AIMP2-DX2 and its uses

ABSTRACT

The present invention relates to a variant of AIMP2 lacking exon 2, named as AIMP2-DX2, which is specifically expressed in cancer cells. The AIMP2-DX2 protein and gene can be successfully used in the development of diagnosis and treatment of cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from Korean Patent Application Nos.10-2004-0097164 and 10-2005-0039073, filed on Nov. 24, 2004 and May 10,2005, respectively in the Korean Intellectual Property Office, thedisclosure of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a variant of AIMP2 lacking exon 2,named as AIMP2-DX2, which is specifically expressed in cancer cells, andits uses in diagnosis and treatment of cancer.

2. Description of the Related Art

Cancer is generally diagnosed by radiography examinations such as X-rayradiography, computed tomography and bronchography or bronchoscopyexamination. However, such methods provide no diagnostic data in termsof cell physiology molecular genetics, while they allow to determineanatomical progress of cancer. Lung and liver cancers are known toexhibit high incidence rate and mortality over the world.

To overcome shortcomings of such conventional examination technologies,a number of markers have been suggested for diagnosing lung or livercancer as described hereunder:

Korean Pat. Appln. No. 10-1998-0038212 relating to the process forevaluating metastasis of lung cancer discloses that local metastaticlung cancer may be assessed by measuring the expression of mitogenactivated protein kinase phosphatase-1 (MKP-1) in lung tissues. WO2004/005891 suggests various disgnostic markers for lung cancer such asAOE372, ATP5D, B4GALT, Ppase, GRP58, GSTM4, P4HB, TPI and UCHL1.Monoclonal antibodies to LCGA have been proposed to diagnose and treatnon-small cell lung carcinoma and ovary cancer as described in U.S. Pat.No. 6,117,981. EP 0804451 discloses a method for diagnosing and treatinglung cancer by use of lung cancer-specific antigen HCAVIII. In addition,U.S. Pat. Nos. 6,746,846 and 6,737,514 and EP 0621480 also discuss lungcancer markers.

Korean Pat. Appln. No. 10-2000-0040609 discloses the early detectionmethod for liver diseases including liver cirrhosis and cancer bymeasuring the level of asialo-glycoproteins in accordance with sandwichassay in which lectins serve as a capture protein and/or probe protein.Korean Pat. Appln. No. 10-2002-0035260 describes that compositionscomprising long-chain fatty-acid-Coenzyme A ligase 4, farnesyldiphosphate synthase, syndecan 2, emopamil-binding protein,preferentially expressed antigen in melanoma and histidine ammonia-lyaseare useful in diagnosing human liver cancer. EP 0334962 suggests thatthe comparison of level of UDP-N-acetyglucosamine with that ofglycoprotein N-acetylglucosamine transferase permits to detect livercancer. Furthermore, EP 0339097 discloses diagnostic methods for livercancer by measuring the level of inhibitors of collagenase in serum,plasma or synovia in a sandwich assay format.

However, markers for lung and liver cancers so far proposed permitrestricted application in the senses that they are also detectable innormal cells. Therefore, assays or diagnostics using such markers aregenerally carried out by comparing their expression levels in normal andcancerous cells, resulting in unreliable and erroneous diagnosis.

Accordingly, there remains a need to propose novel diagnostic makers forlung or live cancer.

Throughout this application, several patents and publications arereferenced and citations are provided in parentheses. The disclosure ofthese patents and publications is incorporated into this application inorder to more fully describe this invention and the state of the art towhich this invention pertains.

SUMMARY OF THE INVENTION

Under such circumstances, the present inventors have made intensiveresearch to develop a novel cancer-specific molecular species and aresult, found that a variant of AIMP2 lacking exon 2, AIMP2-DX2 isspecifically expressed in cancer cells not normal cells and permits todiagnose cancer occurrence in more reliable manner. In addition, thepresent inventors have discovered that antibody, siRNA and antisenseoligonucleotide specific to AIMP2-DX2 allow to effectively treat cancer.

Accordingly, it is an object of this invention to provide a AIMP2-DX2protein with deleted exon 2 region of AIMP2.

It is another object of this invention to provide a nucleic acidmolecule comprising a nucleotide sequence encoding the AIMP2-DX2protein.

It is still another object of this invention to provide a recombinantvector carrying a nucleotide sequence encoding the AIMP2-DX2 protein.

It is further object of this invention to provide a transformant whichis transformed with the recombinant vector carrying a nucleotidesequence encoding the AIMP2-DX2 protein.

It is still further object of this invention to provide an antibodyagainst the AIMP2-DX2 protein.

It is another object of this invention to provide a diagnostic kit forcancer comprising an antibody specific to the AIMP2-DX2 protein.

It is still another object of this invention to provide a method fordiagnosing cancer.

It is further object of this invention to provide an antisenseoligonucleotide which is complementary to a region of an mRNA of theAIMP2-DX2 protein.

It is still further object of this invention to provide a pharmaceuticalcomposition for treating cancer.

It is another object of this invention to provide a method of screeningfor an agent which inhibits the formation of a heterodimer between theAIMP2-DX2 protein and the AIMP2 protein.

It is still another object of this invention to a method of screeningfor an agent which inhibits the expression of the AIMP2-DX2 gene.

Other objects and advantages of the present invention will becomeapparent from the detailed description to follow taken in conjugationwith the appended claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a-1 e represent the functional importance of AIMP2 in TGF-βsignaling and its interaction with Smad2/3. AIMP2^(+/+) and AIMP2^(−/−)MEFs were compared in the effect of TGF-β on cell proliferation (FIG. 1a), colony formation (FIG. 1 b), cell cycle progression (FIG. 1 c), andnuclear translocation of Smad2 and Smad3 (FIG. 1 d). In FIG. 1 a, thecells were incubated with the indicated concentrations (0, 2 and 4ng/ml) of TGF-β1 for 6 hr. Thymidine incorporation in the untreatedcells was taken as 1 and the values are the averages of four independentexperiments. In FIG. 1 b, AIMP2^(+/+) and AIMP2^(−/−) MEFs (14.5 day)were cultivated in the presence of TGF-β (2 ng/ml) for 4 day, fixed withparaformaldehyde, and the colonies were visualized by Giemsa staining.In FIG. 1 c, MEFs were treated with TGF-β for 24 hr, and the portion ofG0/G1 phase cells was determined by flow cytometry (FACS caliubur,Becton Dickinson, US). In FIG. 1 d, MEFs were treated with TGF-β (2ng/ml) for 1 hr. Smad2 and Smad3 were reacted with their specificantibodies and visualized by FITC-conjugated antibody (green). Nucleiwere stained with PI (red). In FIG. 1 e, the interactions of AIMP2 withSmad2 and Smad3 were tested by co-immunoprecipitation with antibodiesagainst Smad2 and Smad3.

FIGS. 2 a-2 f represent the working mechanism of AIMP2 in TGF-βsignaling. In FIG. 2 a, A549 cells were harvested at the indicated timesafter treatment of TGF-β (2 ng/ml) and the extracted proteins wereimmunoprecipitated with anti-Smad2 or Smad3 antibody, andcoprecipitation of AIMP2 was determined with anti-AIMP2 antibody. WCLstands for the Western blots of the proteins of whole cell lysates. InFIG. 2 b, the expression of the TGF-β target genes, p15, p21 and PAI-1was determined by RT-PCR in the control and AIMP2-transfected DU145.FIG. 2 c demonstrates the effect of AIMP2 on the phosphorylation ofSmad2 in AIMP2^(−/−) MEFs. In FIG. 2 d, the different domains of Smad2(Mad-homology domain, MH1, MH2 and linker) were expressed as LexA fusionproteins and tested for the interaction with B42-fused AIMP2 by the bluecolony formation on X-gal-containing yeast medium. In FIG. 2 e, theTGF-β-dependent interaction of Smad2 with TGF-β receptor was determinedby coimmunoprecipitation. MEFs were treated with TGF-β at 37° C. andincubated at 8° C. before immunoprecipitation. The association of TGF-βreceptor with Smad2 was monitored by immunoblotting with anti-TβRIantibody (Santa Cruz biotech). FIG. 2 f shows the time course of thetotal Smad2, phosphorylated Smad2 (p-Smad2), and AIMP2 levels inAIMP2^(+/+) and AIMP2^(−/−) MEFs after TGF-β treatment.

FIGS. 3 a-3 f represents the differential expression of AIMP2 andgeneration of its exon 2-deletion form. The AIMP2 levels were comparedin various cancer cell lines by Western blot analysis (FIG. 3 a) andflow cytometry (FIG. 3 b). A549, NCI-H460, -H322, and H290 are lungcancer cell lines while DU145 and HCT116 are prostate and colon cancercell lines, respectively. In FIG. 3 b, “negative” indicates DU145treated only with secondary antibody. 2000 cells were analyzed for eachcell line. In FIG. 3 c, the AIMP2 transcript level was compared byRT-PCR with different primer pairs. The transcripts spanning exons 3-4and 1-3 were generated by the primer pairs of 6/7 and 1/5 (see FIG. 8b), respectively. GAPDH is a loading control. Note the generation of asmaller AIMP2 transcript from the primer pair for the transcript of exon1-3. This smaller transcript is the alternative splicing form of AIMP2lacking exon 2 (AIMP2-DX2). This transcript was produced only from thelow-AIMP2 cell lines by RT-PCR with the primer AIMP2-DX2-F and the otherone specific to the junction sequence between exon 1 and 3 (AIMP2-DX2-B)(see FIG. 8 b). In FIG. 3 d, TGF-β-dependent induction and nucleartranslocation of AIMP2 were examined by immunofluorescence stainingafter the incubation with TGF-β for 2 hr. In FIG. 3 e, the effect ofTGF-β was compared on the proliferation of the indicated cells by [³H]thymidine incorporation (n=4). In FIG. 3 f, TGF-β-dependent induction ofthe target genes, p21 and PAI-1, was compared by RT-PCR after theincubation with TGF-β for 2 hr.

FIGS. 4 a-4 h demonstrates the Effect of AIMP2-DX2 on the cellularstability of AIMP2. In FIG. 4 a, AIMP2-DX2 was transfected into DU145untreated or treated with TGF-β for 2 hr, and the AIMP2 level wasdetermined by Western blotting. The expression of c-Myc, AIMP2-DX2 andGAPDH (control) was monitored by RT-PCR. In FIG. 4 b, AIMP2-DX2 or emptyvector was transfected into DU145, and its effect on the TGF-β-dependentcell growth inhibition was monitored by thymidine incorporation (n=4).In FIG. 4 c, the interaction between AIMP2-F and AIMP2-DX2 wasdetermined by yeast two hybrid assay as previously described (Rho, S. B.et al., PNAS. USA, 96:4488-93(1999)). In FIG. 4 d, AIMP2-F and AIMP2-DX2were synthesized by in vitro translation in the presence of [³⁵S]methionine, mixed with either GST-AIMP2-F or -CDK2 (control), andprecipitated with glutathione-Sepharose. The precipitated proteins wereseparated by SDS-PAGE and detected by autoradiography. In FIG. 4 e, theinteraction of AIMP2-F or AIMP2-DX2 with FUSE-binding protein (FBP; Kim,M. J. et al., Nat. Genet. 34:330-336:2003)) and Smad2 was determined byyeast two hybrid assay. In FIG. 4 f, the effect of the proteasomeinhibitor, ALLN (50 μM for 4 hr) on the levels of the full-length (F)and AIMP2-DX2 of AIMP2 was monitored in the AIMP2-DX2-generating H322cells by Western blotting with anti-AIMP2 antibody. The AIMP2-DX2 formwas confirmed by its co-migration in gel with its in vitro synthesizedcounterpart. In FIG. 4 g, the increase of AIMP2 by the treatment of ALLN(20 μM for 2 hr) was also shown by immunofluorescence staining withanti-AIMP2 antibody in H322 cells. In FIG. 4 h, myc-tagged AIMP2-DX2 wastransfected to DU145 that were treated with ALLN. Then, AIMP2 wasimmunoprecipitated with anti-AIMP2 antibody, and the ubiquitinated AIMP2molecules were monitored by immunoblotting with anti-ubiquitin antibody(Ubi).

FIGS. 5 a-5 e represent the disruptive effect of AIMP2-DX2 on cellgrowth control and TGF-β signaling. In FIG. 5 a, AIMP2-DX2 (or emptyvector) was transfected into MEFs and monitored its effect on cellgrowth. The cells and colonies were visualized by light microscopy (top)and Giemsa staining (bottom), respectively. In FIG. 5 b, siRNA targetingAIMP2-DX2 (si-DX2) was introduced into H322 and its suppressive effecton the AIMP2-DX2 transcript was determined by RT-PCR (top). si-DX2 didnot affect the full-length AIMP2 transcript as shown by RT-PCR of thetranscript for exon 3-4. The effect of si-DX2 on the phosphorylation ofSmad2 and AIMP2 expression was also determined by Western blotting(bottom). The effect of si-DX2 on the restoration of the TGF-β signalingwas also determined by immunofluorescence staining of p-Smad2 (FIG. 5c), TGF-β-dependent reporter assay under 3TP promoter (FIG. 5 d) andgrowth arrest (FIG. 5 e) using H322 cells. In FIG. 5 c, p-Smad2 andnuclei were stained with FITC-conjugated secondary antibody (green) andPI (red), respectively, 30 min after TGF-β treatment. Notice thatp-Smad2 was increased and nuclear located by the transfection of si-DX2.

FIGS. 6 a-6 c represent the association of AIMP2 with lung cancerformation. In FIG. 6 a, lung tumor formation was monitored at timeinterval after the intraperitoneal administration of benzo-(α)-pyreneinto AIMP2^(+/+) and AIMP2^(+/−) mice. “N” stands for the number of thesacrificed mice. In FIG. 6 b, total RNAs were isolated from the tissues,and subjected to RT-PCR with the AIMP2-DX2-specific primer. Normal andtumor tissues of the same patients (indicated by code number) wereRT-PCR (FIG. 6 c). In FIG. 6 c, the exon 4 region of AIMP2 and GAPDHwere used as control.

FIG. 7 represents the determination of the Smad2 domain involved in theinteraction with AIMP2. We ligated the cDNAs encoding AIMP2 or CDK2 tothe EcoRI and XhoI sites of pGEX4T-1 to express them in E. coli BL21(DE3) as the GST-fusion proteins and purified them following themanufacturer's instruction. The different deletion fragments of Smad2were synthesized by in vitro translation in the presence of [³⁵S]methionine using the TNT coupled translation kit (Promega). The GSTfusion proteins bound to the glutathione Sepharose beads were incubatedwith the [³⁵S] methionine-labeled Smad2 fragments in the binding bufferof PBS buffer (pH 7.4) containing 0.5 mM EDTA, 0.5 mMphenylmethylsulfonylfluoride (PMSF), and 1% Trition X-100. The bindingmixture was incubated overnight 4° C. with rotation and washed fourtimes with the binding buffer containing 0.5% Trition X-100. Afteraddition of the SDS sample buffer, the bound proteins were eluted byboiling and separated by SDS gel electrophoresis. The presence of Smad2fragments was determined by autoradiography.

FIGS. 8 a and 8 b show the exon arrangement of human AIMP2 gene and theprimer locations in AIMP2 cDNA. In FIG. 8 a, the AIMP2 gene is composedof four exons encoding the polypeptides of the indicated size. FIG. 8 bis the schematic representation for the locations of the primers used togenerate the cDNAs spanning different regions of AIMP2 and theirsequences.

FIGS. 9 a-9 d demonstrate reduced expression of AIMP2, and generation ofAIMP2-DX2 in cancer cell lines. In FIG. 9 a, to compare the AIMP2 levelsbetween the AIMP2-DX2-positive and -negative cells, we cultured DU145and H460 cells in one dish, and performed immunofluorescence stainingwith anti-AIMP2 antibody (green). The two cells lines were distinguishedby immunofluorescence staining of p53 (red) since DU145 cells expressp53 at high level due to its mutation, whereas H460 cells containing thewild type p53 maintain it at low level. The cells were treated withTGF-β for 2 hr, and fixed with methanol. In FIG. 9 b, to address theeffect of AIMP2-DX2 on expression of AIMP2, we monitored the AIMP2 levelby flow cytometry. We transfected 2 μg/ml of empty vector or AIMP2-DX2into DU145 cells, and incubated for 24 hr. The cells were then fixedwith 70% ethanol and reacted with anti-AIMP2 antibody, and subsequentlyFITC-conjugated secondary antibody. In FIG. 9 c, the effect of AIMP2-DX2on the TGF-β-dependent cell cycle arrest was compared by flow cytometry.While the portion of the G0/G1 phase cells was increased in DU145 cellstransfected with empty vector, but not in the AIMP2-DX2-transfectedcells. The black and blues lines indicated the cells untreated andtreated with TGF-β, respectively. In FIG. 9 d, we compared the AIMP2levels in H460 cells that were untreated (control) or treated with 10 μMALLN for 2 hr. “Negative” indicates the cells incubated only withFITC-conjugated secondary antibody.

FIG. 10 shows the generation of AIMP2-DX2 and suppression of AIMP2 inlung cancer tissues. Lungs were isolated from the AIMP2^(+/+) andAIMP2^(+/−) mice injected with benzoypyrene, and RNAs were isolated fromeach lung for RT-PCR to determine the generation of AIMP2-DX2. All ofthe lungs generating AIMP2-DX2 showed tumor formation in lung (marked+).

FIG. 11 represents the expression of AIMP2-DX2 and suppression of AIMP2in liver cancer tissues. RT-PCR was performed to determine theexpression of AIMP2-DX2 in liver cancer tissues.

FIG. 12 shows the expression vector carrying the AIMP2-DX2 gene. In FIG.12, the abbreviations, pCMV, BGH pA, f1 ori, neomycin, ampicillin, SV40,SV40 pA, ColE1, T7 and Sp6 denote human cytomegalovirus immediate-earlypromoter, bovine growth hormone polyadenylation signal, f1 replicationorigin, neomycin resistance gene, ampicillin resistance gene, SV40replication origin, SV40 polyadenylation signal, ColE1 replicationorigin, T7 viral promoter and Sp6 viral promoter.

DETAILED DESCRIPTION OF THIS INVETNION

The present inventors elucidated that the genetic disruption of p38(newly designated herein as “AIMP2”) to induce overexpression of c-myccauses neonatal lethality in mice through overproliferation of alveolarepithelial cells and transforming growth factor-β (TGF-β) induces AIMP2expression and promoted its translocation to nuclei for thedownregulation of c-myc (M. J. Kim, et al., Nat. Genet.34:330-336(2003)).

Following the previous research, the present inventors have revealedthat AIMP2 is a novel tumor suppressor, playing a unique role in TGF-βsignaling via interaction with Smad2/3. In addition, we have discoveredthat the aberrant variant of AIMP2 lacking exon II (AIMP2-DX2) isspecifically expressed in cancer cell lines and tissues. The existenceof AIMP2 lacking exon II (AIMP2-DX2) was verified by RT-PCR usingcombinations of AIMP2-specific primers. When the primers were used togenerate AIMP2 cDNA spanning exon 3 and 4, the decrease of AIMP2transcript was not observed in the cells showing the reduced level ofAIMP2 in Western blot analysis. When we used the primers generating thetranscript from exon 1 to 3, we obtained not only the transcript of theexpected size, but also a smaller one. Sequencing analysis of this smalltranscript revealed that it lacks exon 2 encoding 69 amino acid residuesof AIMP2. RT-PCR analysis using the primer targeting to the junctionsequence of exon 1 and 3 showed that the cell lines expressing lowerAIMP2 level generated the smaller transcript, confirming the generationof AIMP2-DX2.

Furthermore, the inventors observed that AIMP2 level was dramaticallyreduced regardless of TGF-β, demonstrating that the generation ofAIMP2-DX2 leads to loss of AIMP2 activity. In addition to this, theintroduction of AIMP2-DX2 elevated the expression of c-myc and relivedthe growth arrest by TGF-β. Surprisingly, we found that AIMP2-DX2 formsa heterodimer with AIMP2 that is ubiquitinated to be rapidly degraded byproteasome-dependent degradation process. Consequently, we are urged toreason that AIMP2-DX2 is closely associated with tumorigenesis byinducing the decrease of AIMP2 level. In vivo study provides additionalevidences to verify that AIMP2-DX2 is strongly related to lung and livercancer formation as well.

It should be noted that p38DX2 described in the priority documents ofthis application, i.e., the Korean Pat. Appln. Nos. 2004-0097164 and2005-0039073 is newly named as AIMP2-DX2.

In one aspect of this invention, there is provided a AIMP2-DX2 proteincomprising a AIMP2 amino acid sequence in which the exon 2 region of theAIMP2 amino acid sequence is deleted, that is specifically expressed incancer cells, in particular, lung and liver cancer cells.

Preferably, the AIMP2-DX2 protein consists of the AIMP2 amino acidsequence in which the exon 2 region of the AIMP2 amino acid sequence isdeleted.

The AIMP2-DX2 protein is a deletion variant of AIMP2 lacking exon 2. Theamino acid sequence of the AIMP2 protein is found in several databases(312aa version: accession Nos. AAC50391.1 and GI:1215669; 320aa version:accession Nos. AAH13630.1, GI:15489023 and BC013630.1 available fromGenBank) and publications (312aa version: Nicolaides, N. C., Kinzler, K.W. and Vogelstein, B. Analysis of the 5′ region of PMS2 revealsheterogeneous transcripts and a novel overlapping gene, Genomics 29(2):329-334(1995); 320 aa version: Generation and initial analysis ofmore than 15,000 full-length human and mouse cDNA sequences, Proc. Natl.Acad. Sci. U.S.A. 99(26): 16899-16903(2002)). The amino acid sequence ofthe AIMP2-DX2 protein comprises, preferably, consists of that of AIMP2lacking exon 2 region as afore-described known sequences. The KoreanPat. Appln. No. 10-2003-0018424 discloses cancer therapy efficacy of theAIMP2 protein, teachings of which are incorporated herein by referencein its entity.

In addition, the AIMP2-DX2 protein includes exon 2-deleted variant ofAIMP2 equivalents, for example, functional equivalents resulting fromsubstitution, deletion, insertion or their combinations of AIMP2 thatexhibit substantially identical activity to the wild type AIMP2, orfunctional derivatives with modifications to alter physical and/orbiochemical properties of the wild type AIMP2 that exhibit substantiallyidentical activity to the wild type AIMP2.

The deletion of exon 2 in AIMP2 as described herein means that the aminoacid sequence spanning exon 2 region in AIMP2 (corresponding to aminoacid 46-114) is partially or wholly deleted to generate a deletionvariant of AIMP2 capable of forming a heterodimer with AIMP2 to inhibitnormal function of AIMP2 and promote degradation of AIMP2. Accordingly,the AIMP2-DX2 protein of this invention may include any variant of AIMP2with whole or partial exon 2 deletion in which exon 1, 3 and/or 4 isnatural or modified by amino acid substitution, deletion or insertion,so long as the variant is able to form a heterodimer with AIMP2 toinhibit normal function of AIMP2. Preferably, the AIMP2-DX2 proteincomprises a whole exon 2 deletion and intact exon 1, 3 and 4. Morepreferably, the AIMP2-DX2 protein comprises, most preferably, consistsof an amino acid sequence of SEQ ID NO:2.

The AIMP2-DX2 protein may comprise its natural-occurring amino acidsequences and variants having modified sequences as well, so long as thevariants retain activity of the AIMP2-DX2 protein described above. Thevariants of the AIMP2-DX2 protein refer to proteins having differentsequences from its natural-occurring amino acid sequence prepared bydeletion, insertion, non-conserved or conserved substitution or theircombinations. The silent alteration of amino acid residues not tosubstantially impair protein activity is well known to one skilled inthe art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York,1979). Such amino acid alteration includes Ala/Ser, Val/Ile, Asp/Glu,Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro,Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly, but not limitedto.

In addition, the AIMP2-DX2 protein may comprise post-translationalmodifications such as phosphorylation, sulfation, acrylation,glycosylation, methylation and farnesylation.

The AIMP2-DX2 protein and its variants may be obtained by the isolationfrom natural sources, synthesis (Merrifleld, J. Amer. Chem. Soc.85:2149-2156(1963)) or recombinant DNA technology (Joseph Sambrook, etal., Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (2001)). Where a recombinantDNA technology is applied, host cells are transformed with an expressionvector carrying a nucleic acid molecule encoding AIMP2-DX2 and thencultured, followed by recovering the AIMP2-DX2 expressed.

As described previously, the AIMP2-DX2 protein is specifically expressedin a variety of cancer cells including breast cancer, large intestinalcancer, lung cancer, small cell lung cancer, stomach cancer, livercancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, heador neck cancer, cutaneous or intraocular melanoma, uterine sarcoma,ovarian cancer, rectal cancer, anal cancer, colon cancer, fallopian tubecarcinoma, endometrial carcinoma, cervical cancer, vulval cancer,vaginal carcinoma, Hodgkin's disease esophageal cancer, small intestinecancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenalcancer, soft tissue tumor, urethral cancer, penile cancer, prostatecancer, bronchogenic cancer, nasopharyngeal cancer, laryngeal cancer,chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidneycancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNStumor, primary CNS lymphoma, bone marrow tumor, brain stem nerve gliomasand pituitary adenoma, in particular, lung and liver cancer tissues,demonstrating that the AIMP2-DX2 protein can serve as a cancerdiagnostic marker.

In another aspect of this invention, there is provided a nucleic acidmolecule comprising a nucleotide sequence encoding the AIMP2-DX2 proteindescribed above.

Preferably, the nucleic acid molecule coding for the AIMP2-DX2 proteincomprises a nucleotide sequence of SEQ ID NO:1. More preferably, thenucleic acid molecule of this invention consists of a nucleotidesequence of SEQ ID NO:1.

It is well understood by the skilled artisan that homologous sequencesdue to codon degeneracy may be encompassed within the nucleic acidmolecule of the present invention, showing at least 60%, preferably 80%,most preferably 90-95% nucleotide similarity to that of SEQ ID NO:1, asmeasured using one of the sequence comparison algorithms. Methods ofalignment of sequences for comparison are well-known in the art. Variousprograms and alignment algorithms are described in: Smith and Waterman,Adv. Appl. Math. 2:482(1981); Needleman and Wunsch, J. Mol. Bio.48:443(1970); Pearson and Lipman, Methods in Mol. Biol. 24:307-31(1988); Higgins and Sharp, Gene 73:237-44(1988); Higgins andSharp, CABIOS 5:151-3(1989); Corpet et al., Nuc. Acids Res.16:10881-90(1988); Huang et al., Comp. Appl. BioSci. 8:155-65(1992); andPearson et al., Meth. Mol. Biol. 24:307-31(1994). The NCBI Basic LocalAlignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol.215:403-10(1990)) is available from several sources, including theNational Center for Biological Information (NBCl, Bethesda, Md.) and onthe Internet, for use in connection with the sequence analysis programsblastp, blasm, blastx, tblastn and tblastx. It can be accessed athttp://www.ncbi.nlm.nih.gov/BLAST/. A description of how to determinesequence identity using this program is available athttp://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

The nucleic acid molecule of this invention may be single- ordouble-chain DNA (cDNA and gDNA) or single-chain RNA (mRNA).

The nucleic acid molecule encoding the AIMP2-DX2 protein may be preparedby the isolation from natural sources, synthesis or recombinant DNAtechnology. The AIMP2-DX2 nucleic acid molecule may be included in asuitable vector to provide the AIMP2-DX2 protein.

In still another aspect of this invention, there is provided arecombinant vector which comprises a nucleic acid molecule comprising anucleotide sequence encoding the AIMP2-DX2 protein.

The term “recombinant vector” used herein refers to a genetic carrier toexpress a protein or RNA of interest in a suitable host cell, comprisinga corresponding foreign sequence operably linked to a nucleic acidexpression control sequence (such as a promoter, signal sequence andarray of transcription factor binding sites).

The term “operably linked” used herein refers to functional linkagebetween a nucleic acid expression control sequence and a second nucleicacid sequence of interest, wherein the expression control sequenceaffects transcription and/or translation of the nucleic acidcorresponding to the second sequence. The vector of this invention maybe constructed according to conventional recombinant DNA technology inwhich site-specific DNA cleavage and ligation are performed usingcommercially available enzymes.

The vector of the present invention includes plasmid, cosmid,bacteriophage and viral vectors, but not limited to. The vector maycomprise expression control elements such as promoter, operator, startand stop codons, polyadenylation signal and enhancer as well as signalor leader sequences for membrane targeting or secretion. The promoterused in vectors may be constitutive or inducible one. Furthermore, thevector may carry a selection marker for selecting host cells harboringthe vector and a replication origin.

The signal sequence in the vector includes, but not limited to, PhoA andOmpA signal sequences for Escherichia host cells, α-amylase andsubtilicin signal sequences for Bacillus host cells, MFα and SUC2 signalsequences for yeast host cells, and insulin, α-interferon and antibodymolecule signal sequences for animal host cells.

In further aspect of this invention, there is provided a transformantwhich is transformed with the recombinant vector of this inventiondescribed above.

The transformation may be carried out according to any known approachfor transforming nucleic acid molecules into organism, cell, tissue ororgan, including electroporation, protoplasm fusion, CaPO₄precipitation, CaCl₂ precipitation, agitation using silicon carbamidefiber, Agrobacterium-mediated transformation, PEG, dextran sulfate andlipofectamine, but not limited to. A suitable transformation method maybe selected based on the type of host cells.

A suitable host cell is generally decided in considering the expressionlevel and post-translation modification. Host cells include, but notlimited to, prokaryotic cells such as Escherichia coli, Bacillussubtilis, Streptomyces, Pseudomonas, Proteus mirabilis andStaphylococcus, fungi (e.g., Aspergillus), yeast (e.g., Pichia pastoris,Saccharomyces cerevisiae, Schizosaccharomyces and Neurospora crassa),insect cells, plant cells and mammalian cells.

In still further aspect of this invention, there is provided a processfor preparing the AIMP2-DX2 protein by culturing transformed cellsdescribed previously.

The culturing is carried out by conventional methods known to thoseskilled in the art under conditions suitable to express the AIMP2-DX2protein of interest.

The AIMP2-DX2 protein expressed may be purified by conventional methods,for example, salting out (e.g., ammonium sulfate and sodium phosphateprecipitation), solvent precipitation (e.g., protein fractionationprecipitation using acetone or ethanol), dialysis, gel filtration, ionexchange, reverse-phase column chromatography, ultrafiltration or theircombinations (Sambrook et al., Molecular Cloning: A Laboratory Manual,3rd Ed., Cold Spring Harbor Laboratory Press (2001); and Deutscher, M.,Guide to Protein Purification Methods, Enzymology, vol. 182. AcademicPress. Inc., San Diego, Calif. (1990)).

The term “cancer marker” used herein refers to a substance that providesinformation to evaluate cancer likelihood, occurrence or development byexamining qualitatively or quantitatively its expression in tissues orcells. Preferably, the term means an organic biomolecule (e.g., protein,DNA and RNA) that is expressed in cancer cells in a different patternfrom normal cells. The AIMP2-DX2 gene or protein of this invention isspecifically expressed in cancer cells but not in normal cells,permitting to accurately diagnose cancer (preferably, lung and livercancer). Preferably, the cancer diagnosis using AIMP2-DX2 expression isperformed in mRNA and/or protein level.

In another aspect of this invention, there is provided an antibodyspecific to the AIMP2-DX2 protein.

The term “antibody” used herein means a protein molecule specificallydirected toward an antigenic site. The antibody of this invention refersto antibodies to specifically recognize AIMP2-DX2 with discriminatingAIMP2, including polyclonal and monoclonal antibodies.

Antibodies against the novel protein, AIMP2-DX2, may be prepared inaccordance with conventional technologies known to one skilled in theart.

Polyclonal antibodies may be prepared according to known processes inwhich the AIMP2-DX2 protein as an immunogen is injected into animals andthen antiserum is collected. Immunized animals include, but not limitedto, goat, rabbit, sheep, monkey, horse, pig, cattle and dog.

Monoclonal antibodies may be prepared in accordance with a fusion method(Kohler and Milstein, European Journal of Immunology, 6:511-519(1976)),a recombinant DNA method (U.S. Pat. No. 4,816,56) or a phage antibodylibrary (Clackson et al, Nature, 352:624-628(1991); and Marks et al, J.Mol. Biol., 222:58, 1-597(1991)).

Antibodies against the AIMP2-DX2 protein may be an intact immunoglobulinmolecule or its fragments containing antigen-binding site such as F(v),Fab, Fab′ and F(ab′)2.

The antibodies of this invention specific to AIMP2-DX2 permit todiagnose cancer (preferably, lung and liver cancer) as well as to treatcancer (preferably, lung and liver cancer) by suppressing the activityof AIMP2-DX2. Where the antibodies are used as a therapeutic agent, theymay be coupled to conventional therapeutic agents in direct or indirect(through a linker) manner.

The therapeutic agent coupled to the antibodies of this inventionincludes, but not limited to, radionuclide (e.g., 131I, 90Y, 105Rh,47Sc, 67Cu, 212Bi, 211At, 67Ga, 125I, 186Re, 188Re, 177Lu, 153Sm, 123Iand 111In), drug (e.g., methotrexate and adriamycin), lymphokine(interferon), toxin (ricin, abrin and diphtheria) and heterofunctionalantibody that forms a complex with other antibody to posses abi-functional binding capacity both to cancer cell and effector cell(e.g., T killer cell).

The antibody of this invention may be administered per se or in the formof a pharmaceutical composition.

The pharmaceutical composition comprising antibody may be formulatedwith a pharmaceutically acceptable carrier. The form of thepharmaceutical composition varies depending on the administration mode.Typically, the composition comprises one of surfactants for facilitatingtransmembrane delivery. Such surfactant includes steroid-derivedcompounds, cationic lipids such asN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),cholesterol hemisuccinate and phosphatidyl glycerol.

The pharmaceutical composition comprising the antibody of this inventionis administered in a pharmaceutically effective amount to treat canceror prevent cancer metastasis. The pharmaceutical composition may beadministered in a single or multiple dosing regimen. The administrationmode of the pharmaceutical composition includes parenteral,subcutaneous, intraperitoneal, intrapulmonary, intranasal, and localadministration. Parenteral administration includes subcutaneous,intradermal, intramuscular, intravenous, intrabursa, intrasternal,intrathecal and intraperitoneal injection. The pharmaceuticalcomposition is generally formulated in a pH range of 4-8 for antibodystability (chemical and physical stability) and safety. In addition, thepharmaceutical composition may be formulated in an oral dosing form.Typical dose is optimized using standard clinical techniques.

Furthermore, the antibody of this invention may be administered in aform of nucleic acid molecule to induce in vivo production of antibody(WO 96/07321).

In still another aspect of this invention, there is provided adiagnostic kit for cancer, which comprises an antibody specific to theAIMP2-DX2 protein.

The cancer diagnosis kit of this invention may comprise antibodyspecific to AIMP2-DX2 as well as general instruments and reagents forimmunoassay including carrier, detectable signal-generating label,dissolving agent, washing agent, buffer and stabilizer. Where an enzymeis used as a label, its substrate and reaction quencher may be included.Non-limiting examples of carrier include soluble carriers, for example,physiologically acceptable buffer known in the art (e.g. PBS), insolublecarriers, for example, polystyrene, polyethylene, polypropylene,polyester, polyacrylonitrile, fluorine resin, cross-linked dextran,polysaccharides, polymers such as magnetic microparticles made of latexcoated with a metal, paper, glass, metals, agarose and combinationsthereof.

Non-limiting examples of the assay system useful in the cancer diagnosiskit of the present invention include ELISA plates, dip-stick devices,immunochromatography test strips and radial partition immunoassy devicesand flow-through devices.

In further aspect of this invention, there is provided a method fordiagnosing cancer, which comprises the steps of: (a) providing a sampleto be assayed; and (b) detecting in the sample an expression of anucleotide sequence encoding the AIMP2-DX2 protein of claim 1, whereinthe detection of the expression of the nucleotide sequence encoding theAIMP2-DX2 protein is indicative of cancer.

The sample used in the present invention includes any biological samplesuch as tissue, cell, whole blood, serum, plasma, saliva, semen, urine,synovia and spinal fluid and may be pretreated for assay.

The present method may be carried out at protein or mRNA level. Where itis performed to detect the AIMP2-DX2 protein, antibodies to specificallyrecognize the AIMP2-DX2 protein are used and the detection is carriedout by contacting the sample to the antibody specific to the AIMP2-DX2protein and evaluating a formation of antigen-antibody complex. Theevaluation on antigen-antibody complex formation may be carried outusing immunohistochemical staining, radioimmuno assay (RIA),enzyme-linked immunosorbent assay (ELISA), Western blotting,immunoprecipitation assay, immunodiffusion assay, complement fixationassay, FACS and protein chip assay. The evaluation on antigen-antibodycomplex formation may be performed qualitatively or quantitatively, inparticular, by measuring signal from detection label.

The label to generate measurable signal for antigen-antibody complexformation includes, but not limited to, enzyme, fluorophore, ligand,luminophore, microparticle, redox molecules and radioisotopes. Theenzymatic label includes, but not limited to, β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, peroxidase, alkalinephosphatase, acetylcholinesterase, glucose oxidase, hexokinase, GDPase,RNase, luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase,aspartate aminotransferase, phosphenolpyruvate decarboxylase,β-lactamase. The fluorescent label includes, but not limited to,fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin,allophysocyanin, o-phthalate and fluorescamine. The ligand serving as alabel includes, but not limited to, biotin derivatives. Non-limitingexamples of the luminescent label includes acridinium ester, luciferinand luciferase. Microparticles as label include colloidal gold andcolored latex, but not limited to. Redox molecules for labeling includeferrocene, lutenium complex compound, viologen, quinone, Ti ion, Cs ion,diimide, 1,4-benzoquinone, hydroquinone, K₄ W(CN)₈, [Os (bpy)₃]²⁺,[Ru(bpy)₃]²⁺ and [Mo(CN)₈]⁴⁻, but not limited to. The radioisotopesincludes ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I,¹³¹I and ¹⁸⁶Re, but not limited to.

Where the present method is performed to detect the AIMP2-DX2 mRNA, thedetection step may be carried out by an amplification reaction or ahybridization reaction well-known in the art.

The phrase “detection of the AIMP2-DX2 mRNA” used herein is intended torefer to analyze the existence or amount of the AIMP2-DX2 mRNA as cancerdiagnosis marker in cells by use of primer or probe specificallyhybridized with the AIMP2-DX2 mRNA.

The term “primer” used herein means an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, i.e., in the presence of four different nucleosidetriphosphates and a thermostable enzyme in an appropriate buffer and ata suitable temperature.

The term “probe” used herein refers to a linear oligomer of natural ormodified monomers or linkages, including deoxyribonucleotides,ribonucleotides and the like, which is capable of specificallyhybridizing with a target nucleotide sequence, whether occurringnaturally or produced synthetically. The probe used in the presentmethod may be prepared in the form of oligonucleotide probe,single-stranded DNA probe, double-stranded DNA probe and RNA probe. Itmay be labeled with biotin, FITC, rhodamine, DIG and radioisotopes.

The method to detect the AIMP2-DX2 mRNA using either primer or probeincludes, but not limited to, DNA sequencing, RT-PCR (reversetranscription-polymerase chain reaction), primer extension method(Nikiforov, T. T. et al., Nucl Acids Res 22, 4167-4175(1994)),oligonucleotide ligation analysis (OLA) (Nickerson, D. A. et al., ProNat Acad Sci USA, 87, 8923-8927(1990)), allele-specific PCR (Rust, S. etal., Nucl Acids Res, 6, 3623-3629(1993)), RNase mismatch cleavage (MyersR. M. et al., Science, 230, 1242-1246(1985)), single strand conformationpolymorphism (SSCP; Orita M. et al., Pro Nat Acad Sci USA, 86,2766-2770(1989)), simultaneous analysis of SSCP and heteroduplex (Lee etal., Mol Cells, 5:668-672(1995)), denaturation gradient gelelectrophoresis (DGGE; Cariello N F. et al., Am J Hum Genet, 42,726-734(1988)) and denaturing high performance liquid chromatography(D-HPLC, Underhill Pa. et al., Genome Res, 7, 996-1005(1997)).

Preferably, the method by amplification reaction is carried out byRT-PCR using a primer capable of differentiating an mRNA of AIMP2-DX2from an mRNA of AIMP2. RT-PCR process suggested by P. Seeburg (1986) forRNA research involves PCR amplification of cDNA obtained from mRNAreverse transcription. For amplification, a primer pair specificallyannealed to AIMP2-DX2 is used. Preferably, the primer is designed togenerate two different sized bands in electrophoresis in which one isspecific to the AIMP2 mRNA and the other to AIMP2-DX2 mRNA.Alternatively, the primer is designed to generate only electrophoresisband specific to AIMP2-DX2 mRNA. The primer pair to generate twodifferent sized bands for the AIMP2 mRNA and AIMP2-DX2 mRNA is preparedto amplify a region corresponding to exon 2. The nucleotide sequence ofsuch primers is not limited; most preferably, a primer set consisting ofSEQ ID NOs:5 and 6. To observe only one electrophoresis band specific toAIMP2-DX2 mRNA, one of primers is designed to comprise the junctionsequence between C-terminal of exon 1 and N-terminal of exon 3. InExamples described below, the primer of SEQ ID NO:8 annealed to thejunction sequence is used together with the primer of SEQ ID NO:7 forRT-PCR. The RT-PCR analysis is convenient in the senses that cancerdiagnosis is accomplished by observing the electrophoresis band patternto evaluate expression of the AIMP2-DX2 mRNA.

The present method may be carried out in accordance with hybridizationreaction using suitable probes.

The stringent conditions of nucleic acid hybridization suitable forforming such double stranded structures are described by JosephSambrook, et al., Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) and Haymes, B.D., et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press,Washington, D.C. (1985). As used herein the term “stringent condition”refers to the conditions of temperature, ionic strength (bufferconcentration), and the presence of other compounds such as organicsolvents, under which hybridization or annealing is conducted. Asunderstood by those of skill in the art, the stringent conditions aresequence dependent and are different under different environmentalparameters. Longer sequences hybridize or anneal specifically at highertemperatures.

The probes used in the hybridization reaction have a AIMP2-DX2 specificnucleotide sequence which is not found in AIMP2. Preferably, the probesare designed to comprise the junction sequence between exons 1 and 3,most preferably, having the nucleotide sequence of SEQ ID NO:8.

The present method is very useful in diagnosing a variety of cancerincluding breast cancer, large intestinal cancer, lung cancer, smallcell lung cancer, stomach cancer, liver cancer, blood cancer, bonecancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneousor intraocular melanoma, uterine sarcoma, ovarian cancer, rectal cancer,anal cancer, colon cancer, fallopian tube carcinoma, endometrialcarcinoma, cervical cancer, vulval cancer, vaginal carcinoma, Hodgkin'sdisease esophageal cancer, small intestine cancer, endocrine cancer,thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue tumor,urethral cancer, penile cancer, prostate cancer, bronchogenic cancer,nasopharyngeal cancer, laryngeal cancer, chronic or acute leukemia,lymphocytic lymphoma, bladder cancer, kidney cancer, ureter cancer,renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNSlymphoma, bone marrow tumor, brain stem nerve gliomas and pituitaryadenoma. More preferably, the present method is used for diagnosing lungcancer and liver cancer, most preferably, lung cancer.

In still further aspect of this invention, there is provided a siRNA(small interfering RNA) molecule which comprise a nucleotide sequencecomplementary to a region of an mRNA of the AIMP2-DX2 protein.

The term “siRNA” used herein refers to a short RNA duplex to induce RNAi(RNA interference) phenomenon through mRNA cleavage. The siRNA consistsof a sense RNA strand corresponding to target mRNA and an antisense RNAstrand complementary to target mRNA. siRNA to inhibit expression of atarget gene provides effective gene knock-down method or gene therapymethod.

The siRNA of this invention is not restricted to a RNA duplex of whichtwo strands are completely paired and may comprise non-paired portionsuch as mismatched portion with non-complementary bases and bulge withno opposite bases. The overall length of the siRNA is 10-100nucleotides, preferably, 15-80 nucleotides, and more preferably, 20-70nucleotides. The siRNA may comprise either blunt or cohesive end so longas it enables to silent the AIMP2-DX2 expression due to RNAi effect. Thecohesive end may be prepared in 3′-end overhanging structure or 5′-endoverhanging structure. The overhanging bases are not limited in itslength, for example, 1-8 nucleotides, preferably, 2-6 nucleotides. Theoverall length as described herein is expressed as the total of lengthof central double-stranded portion and terminal single-strandedoverhanging portion. Furthermore, as long as AIMP2-DX2 siRNA is able tomaintain its gene silencing effect on the target gene, it may contain alow molecular weight RNA (which may be a natural RNA molecule such astRNA, rRNA or viral RNA, or an artificial RNA molecule), for example, inthe overhanging portion at its either end. It is not necessary that bothends of AIMP2-DX2 siRNA have a cleavage structure. The AIMP2-DX2 siRNAof this invention may comprise a stem-loop structure in which either end(head or tail) is connected via a linker. The length of the linker isnot limited unless it impairs base pairing in stem structure.

The term “specific” used herein in conjunction with siRNA is intended toexpress the inhibition of target gene expression with no influence onother genes. The siRNA of this invention is specific to the AIMP2-DXgene.

It is preferred that the siRNA of this invention comprise a sense strandcontaining a corresponding sequence to a junction sequence between exons1 and 3 and an antisense strand containing a complementary sequence.

The phrase “inhibition of gene expression” means that the level of mRNAand/or protein generated from the target gene is quenched or reduced,which is induced by RNA interference via occurrence of mRNA cleavage.

The siRNA of this invention may be synthesized in vitro and thenintroduced into cells via transfection. In addition, it may betransfected into cells in the form of siRNA expression vector orPCR-derived siRNA expression cassette. Suitable preparation andtransfection methods may be determined in considering the experiment aimand target gene function.

The sequences and length of the siRNA are not limited as long as itenables to suppress the AIMP2-DX2 expression. 3 illustrative siRNAexpression vectors to silence AIMP2-DX2 are found in Examples describedhereunder, suppressing cellular level of AIMP2-DX2 and restoring AIMP2function and TGF-β signal transduction.

The preferable siRNA of this invention comprises a correspondingsequence to the junction sequence between exons 1 and 3 of the AIMP2-DX2mRNA. More preferably, the siRNA of this invention is a RNA duplexdescribed as (i) No.3 siRNA consisting of two RNA molecules expressedfrom nucleotide sequences of SEQ ID NOs:9 and 10, (ii) No.4 siRNAconsisting of two RNA molecules expressed from nucleotide sequences ofSEQ ID NOs:11 and 12, or (iii) No.5 siRNA consisting of two RNAmolecules expressed from nucleotide sequences of SEQ ID NOs:13 and 14.The siRNA consisting of two RNA molecules expressed from nucleotidesequences of SEQ ID NOs:11 and 12 is most preferred. The single or mixedtype of siRNA molecules may be used.

In another aspect of this invention, there is provided an antisenseoligonucleotide which is complementary to a region of an mRNA of theAIMP2-DX2 protein.

The term “antisense oligonucleotide” used herein is intended to refer tonucleic acids, preferably, DNA, RNA or its derivatives, that arecomplementary to the base sequences of a target mRNA, characterized inthat they binds to the target mRNA and interfere its translation toprotein. The antisense oligonucleotide of this invention means DNA orRNA sequences complementary and binding to AIMP2-DX2 mRNA, that are ableto inhibit translation, translocation, maturation or other biologicalfunctions of AIMP2-DX2 mRNA. The antisense nucleic acid is 6-100,preferably, 8-60, more preferably, 10-40 nucleotides in length.

The antisense oligonucleotide may at lease one modification in its base,sugar or backbone for its higher inhibition efficacy (De Mesmaeker etal., Curr Opin Struct Biol., 5(3):343-55(1995)). The modified nucleicacid backbone comprises phosphorothioate, phosphotriester, methylphosphonate, short chain alkyl or cycloalkyl intersugar linkages orshort chain heteroatomic or heterocyclic intersugar linkages. Theantisense oligonucleotide may also contain one or more substituted sugarmoieties. The antisense nucleic acid may include one or more modifiedbases, for example, hypoxanthine, 6-methyladenine, 5-me pyrimidines(particularly, 5-methylcytosine), 5-hydroxymethylcytosine (HMC),glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases,e.g., 2-aminoadenine, 2-(methylamino)adenine,2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or otherheterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine,5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine,N⁶(6-aminohexyl)adenine and 2,6-diaminopurine. Another modification ofthe oligonucleotides of the invention involves chemically linking to theoligonucleotide one or more moieties or conjugates which enhance theactivity or cellular uptake of the oligonucleotide. Such moietiesinclude but are not limited to lipid moieties such as a cholesterolmoiety, a cholesteryl moiety (Letsinger et al., Proc. Natl. Acad. Sci.USA, 86:6553(1989)), cholic acid (Manoharan et al. Bioorg. Med. Chem.Let., 4:1053(1994)), a thioether, e.g., hexyl-S-tritylthiol (Manoharanet al. Ann. N.Y. Acad. Sci., 660:306(1992); Manoharan et al. Bioorg.Med. Chem. Let., 3: 2765(1993)), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 20:533(1992)), an aliphatic chain, e.g., dodecandiolor undecyl residues (Saison-Behmoaras et al. EMBO J., 10:111(1991);Kabanov et al. FEBS Lett., 259:327(1990); Svinarchuk et al. Biochimie,75:49(1993), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al. Tetrahedron Lett., 36:3651(1995); Shea et al. Nucl.Acids Res., 18:3777(1990)), a polyamine or a polyethylene glycol chain(Manoharan et al. Nucleosides & Nucleotides, 14:969(1995)), oradamantane acetic acid (Manoharan et al. Tetrahedron Lett., 36:3651(1995)). Oligonucleotides comprising lipophilic moieties, andmethods for preparing such oligonucleotides are known in the art, forexample, U.S. Pat. Nos. 5,138,045, 5,218,105 and 5,459,255. Themodifications described above enhance stability against nucleasedegradation and increase affinity of the antisense oligonucleotidetoward its target mRNA.

It is preferred that the antisense oligonucleotide specific to AIMP2-DX2comprises a complementary sequence to a junction region between exons 1and 3 of AIMP2-DX2 mRNA.

The antisense RNA molecule is conventionally synthesized in vitro andthen transmitted to cells. In addition, it is intracellularly producedby transcription from foreign sequence. In vitro synthesis involves RNApolymerase I. In vivo transcription for preparing antisense RNA usesvector having origin of recognition region (MCS) in oppositeorientation. The antisense RNA preferably comprises a translation stopcodon for inhibiting translation to peptide.

In another aspect of this invention, there is provided a pharmaceuticalcomposition for treating cancer, which comprises (a) the antisenseoligonucleotide or siRNA specific to AIMP2-DX2 mRNA as an activeingredient; and (b) a pharmaceutically acceptable carrier.

In still another aspect of this invention, there is provided a methodfor treating cancer in a patient, which comprises administrating intothe patient a pharmaceutical composition (a) the antisenseoligonucleotide or siRNA specific to AIMP2-DX2 mRNA as an activeingredient; and (b) a pharmaceutically acceptable carrier.

The pharmaceutical composition comprising at least one of AIMP2-DX2siRNAs or antisense oligonucleotides may contain additional agent tosuppress tumor cell proliferation and to facilitate the transduction ofsiRNA or antisense nucleic acid, for example, liposome (U.S. Pat. Nos.4,897,355, 4,394,448, 4,235,871, 4,231,877, 4,224,179, 4,753,788,4,673,567, 4,247,411 and 4,814,270), or lipophilic carrier includingsterols such as cholesterol, cholate and deoxycholic acid. In addition,the siRNA or antisense nucleic acid is conjugated to cell-adsorbingpeptides such as peptide hormones, antigens and peptide toxins(Haralambid et al, WO 89/03849; Lebleu et al., EP 0263740).

Where the pharmaceutical composition is formulated for oraladministration, it may contain binder, lubricant, disintegrator,diluent, solubilizer, dispersing agent, stabilizer, suspending agent,pigment and sweetener. Where the pharmaceutical composition isformulated for injection, it may contain buffer, preservative,solubilizer, tonicity agent and stabilizer. For topical administration,the pharmaceutical composition may contain substrate, diluent, lubricantand preservative. The formulation of the pharmaceutical composition maybe prepared by formulating with pharmaceutically acceptable carriersdescribed above. For oral administration, the pharmaceutical compositionmay be in the form of tablet, troche, capsule, elixir, suspension, syrupand wafer. The injectable composition may be formulated in unit dosageample or multi dosage form.

The correct dosage of the pharmaceutical compositions of this inventioncomprising AIMP2-DX2 siRNA or antisense oligonucleotide will be variedaccording to the particular formulation, the mode of application, age,body weight and sex of the patient, diet, time of administration,condition of the patient, drug combinations, reaction sensitivities andseverity of the disease. It is understood that the ordinary skilledphysician will readily be able to determine and prescribe a correctdosage of this pharmaceutical compositions.

The administration mode of the pharmaceutical composition includes oraland parenteral such as subcutaneous, intradermal, intramuscular,intravenous, intrabursa, intrasternal, intrathecal and intraperitonealinjections.

In further aspect of this invention, there is provided a method ofscreening for an agent which inhibits the formation of a heterodimerbetween the AIMP2-DX2 protein of claim 1 and the AIMP2 protein,comprising the steps of: (a) contacting a test substance to acomposition which comprises the AIMP2-DX2 protein and the AIMP2 protein;and (b) determining whether the test substance inhibits the heterodimerformation between the AIMP2-DX2 protein and the AIMP2 protein, whereinthe test substance to inhibit the heterodimer formation between theAIMP2-DX2 protein and the AIMP2 protein is evaluated as an anticanceragent.

The formation of the heterodimer between the AIMP2-DX2 protein and AIMP2protein is associated with cancer as demonstrated in Examples describedhereunder. Therefore, a substance capable of inhibiting the heterodimerformation is evaluated as a candidate for anticancer agent.

According to a preferred embodiment, the instant method is performed bya yeast-two-hybrid assay and in vitro pull-down assay.

Where the present method is carried out by yeast-two-hybrid assayformat, the composition comprising the AIMP2-DX2 protein and the AIMP2protein is a cell harboring the respective gene.

In a yeast two-hybrid assay, the AIMP2-DX2 protein and AIMP2 protein canbe used as either “bait” or “prey” (see, e.g., U.S. Pat. No. 5,283,317;Zervos et al., Cell 72, 223-232(1993); Maduraetal., J. Biol. Chem. 268,12046-12054(1993); Bartel et al., BioTechniques 14, 920-924(1993);Iwabuchi et al., Oncogene 8, 1693-1696(1993); and Brent WO 94/10300), toidentify substances which inhibits the interaction of AIMP2-DX2 withAIMP2 to form a heterodimer. The two-hybrid system is based on themodular nature of most transcription factors, which consist of separableDNA-binding and activation domains. Briefly, the assay utilizes twodifferent DNA constructs. For example, in one construct, polynucleotideencoding the AIMP2-DX2 protein can be fused to a polynucleotide encodingthe DNA binding domain of a known transcription factor (e.g. Lex). Inthe other construct a DNA sequence that encodes the AIMP2 protein can befused to a polynucleotide that codes for the activation domain of theknown transcription factor (e.g. B42).

If the test substance treated to cells expressing the two-hybrid systemis able to inhibit the interaction between the AIMP2-DX2 protein andAIMP2 protein, the DNA-binding and activation domains of thetranscription factor are not brought into close proximity. Thisproximity allows transcription of a reporter gene (e.g. lac Z), which isoperably linked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be easilydetected.

In an in vitro pull-down assay format, the AIMP2-DX2 gene and AIMP2 genecan be used as either bait or prey. For example, in one construct forbait, the AIMP2 protein is fused to a protein that allows the AIMP2protein to be bound to a solid support. For example,glutathione-S-transferase (GST) fusion proteins can be adsorbed ontoglutathione Sepharose beads or glutathione derivatized microtiterplates. In the other construct for prey, the AIMP2-DX2 protein issynthesized by in vitro translation system (e.g., reticulocyte lysate)using ³⁵S. The radioactive AIMP2-DX2 protein is added to the GST-AIMP2protein bound to glutathione Sepharose beads together with the additionof the test substance. The reaction mixture is washed and the proteinsbound to the Sepharose beads are eluted, followed by electrophoresis.

If the test substance added to the reaction mixture is able to inhibitthe interaction between the AIMP2-DX2 protein and AIMP2 protein, theelectrophoresis result shows no band.

In still further aspect of this invention, there is provided a method ofscreening for an agent which inhibits the expression of the AIMP2-DX2gene, comprising the steps of: (a) contacting a test substance to cellswhich express the AIMP2-DX2 gene; and (b) determining whether the testsubstance inhibits the expression of the AIMP2-DX2 gene, wherein thetest substance to inhibit the expression of the AIMP2-DX2 gene isevaluated as an anticancer agent.

In the present method, the expression of the AIMP2-DX2 gene is assessedat mRNA or protein level. Where the present method is carried out todetect the AIMP2-DX2 mRNA expressed, it is preferably performed byRT-PCR using AIMP2-DX2 specific primers described hereinabove. Where thepresent method is carried out to detect the AIMP2-DX2 protein expressed,it is preferably performed by a variety of immunoassay processes usingAIMP2-DX2 specific antibodies as described above.

The following specific examples are intended to be illustrative of theinvention and should not be construed as limiting the scope of theinvention as defined by appended claims.

EXAMPLES

Methods

Cell Culture, Chemicals and Cell Cycle Measurement

Cells were maintained in RPMI-1640 containing 10% FBS. Mouse embryonicfibroblasts (MEFs) were isolated from 12.5-14.5 day embryos andcultivated in DMEM (Dulbecco's Modified Eagle Medium) containing 20%FBS. To evaluate the effect of TGF-β on cell cycle, cells were incubatedwith 2 ng/ml TGF-β in serum-free or 1% FBS-containing medium for 24 hrand harvested for FACS analysis. Cell proliferation was also determinedby [³H] thymidine incorporation. Cells were incubated in serum-freemedium with or without TGF-β for 20 hr, and then in the presence of 1μCi/ml of [³H] thymidine for 4 hr. The incorporated thymidine wasquantified by liquid scintillation counting as previously described(Kim, M. J. et al., Nat. Genet. 34, 330-336(2003)). TGF-β was purchasedfrom R&D system, and anti-Smad2 and anti-Smad4 antibodies from SantaCruz.

Immunoblotting and Immunoprecipitation

Cells were treated with TGF-β for the indicated times and proteins wereextracted with protease-containing RIPA buffer (1% Nonidet P-40, 0.5%sodium deoxycholate, 0.1% SDS), separated by 10-12% SDS-PAGE, andimmunoblotted with the specific antibodies using ECL system (Santa cruzbiotech). For immunoprecipitation, the cell lysates were cleared bypre-incubation with IgG (Pierce) and agarose-conjugated protein A(Invitrogen). After centrifugation, the supernatants were incubated withthe specific antibody, and agarose-conjugated protein A for 2 hr. Afterwashing with ice-cold PBS twice and RIPA once, the bound proteins wereprecipitated with the specific antibody, eluted and subjected to Westernblot analysis.

RT-PCR

The total RNAs were isolated following the protocol of the manufacturer(Qiagen). Briefly, the freshly prepared tissues (3×3×3 mm) were choppedinto small pieces, mixed with 350 μl lysis buffer and homogenized usinghomogenizer or syringe. After adding 350 μl of 70% ethanol, the lysateswere inverted several times, loaded onto column, centrifuged at 13,000RPM for 15 sec. After washing the column with wash buffer twice, RNAswere eluted with 40 μl RNase-free DW. For reverse transcription, 1 μg ofthe isolated RNA was used as a template with the AIMP2-specific primer(FIG. 8 b). After the reaction, the mixture was diluted 3 fold with DWand 1 μl of its aliquot was used for 30 μl PCR reaction containing 0.5μl dNTP (2.5 mM each), 0.5 μl of the indicated primers (each 10 μM), 1.5μl DMSO and 0.1 μl Taq polymerase (5 U/μl).

Suppression of AIMP2-DX2 with si-RNA

To suppress the expression of AIMP2-DX2, we designed the siRNA againstAIMP2-DX2 with the sequence ofTCGAGCTGGCCACGTGCAGGATTACGAGTACTGGTAATCCTGCACGTGGCCAGCTTTT (underlinedregions are matched to the AIMP2-DX2 sequence) and cloned it into 16IMT-700 vector system (Imgenex) using SalI and XbaI, followed by DNAsequencing for confirming the cloned sequence. H322 cells (ATCC, humanlung epithelial carcinoma) were transfected with 2 μg of si-AIMP2-DX2expression vector. Following 3-hr incubation with DNA-liposome complexin 1 ml serum-free media, H322 cells were treated with 1 ml RPMI-1640containing 20% FBS. Then, cells were incubated in serum-free medium withor without TGF-β for 20 hr.

Yeast Two-Hybrid Analysis

The cDNAs encoding human AIMP2 and Smad2 (and its deletion fragments)were obtained by PCR using specific primers. The PCR products of Smad2and AIMP2 were digested with EcoRI and XhoI, and ligated into the samesites of pEG202 (LexA) and pJG4-5 (B42), respectively (Gyuris, J et al.,a human G1 and S phase protein phosphatase that associates with Cdk2.Cell 75, 791-803(1993)). The interactions between Lex-Smad2 fragmentsand B42-AIMP2 were analyzed for their ability to grow on yeast mediumcontaining X-gal as previously described (Rho, S. B. et al., Proc NatlAcad Sci USA 96, 4488-93. (1999)).

In Vitro Pull-Down Assay

AIMP2, AIMP2-DX2 and CDK2 were expressed as GST fusion proteins andimmobilized to glutathione-Sepharose 4B (Pharmacia). The cDNA fragmentsencoding the AIMP2 and AIMP2-DX2 were obtained by PCR using specificprimers and cloned into pET-28a vector for in vitro transcription andtranslation (Promega). Aliquots (10 μl) of TNT products were incubatedwith 5 μg of GST-AIMP2, -AIMP2-DX2 and -CDK2 immobilized on the beads in100 μl of PBS containing 0.5% Triton X-100, 0.5 mM EDTA, and 0.5 mMphenylmethylsulfonyl fluoride. The beads were vigorously washed with thebinding buffer, and the bound proteins were eluted, resolved bySDS-PAGE, and determined by autoradiography.

Construction of Cells Stably Generating AIMP2-DX2

The wild type MEFs were transfected with pcDNA-AIMP2-DX2 or pcDNA(Invitrogen) itself and the transfectants were selected in DMEM mediumcontaining 400 μg/ml G418. After removing the untransfected cells, thetransfectants were cultured in the normal medium without G418 for 3days, fixed with 2% PFA and stained with Giemsa.

Immunostaining and Histological Analysis

The frozen tissue slides were fixed with 2% paraformaldehyde and washedwith ice cold PBS. After blocking and permeablization with PBScontaining 0.2% Triton X-100 (PBST) and 1% BSA, the slides wereincubated with anti-AIMP2 antibody for 2 hr. After washing with PBS,they were also incubated with anti-rabbit goat IgG-FITC (Pierce) andpropidium iodide (50 μg/ml, Sigma) for 1 hr, washed with PBS, mounted,and observed under a confocal microscopy (μ Radiance, Bio-Rad).

Construction of AIMP2-DX2 Expression Vector

To construct the AIMP2-DX2 expressing vector, cDNA of AIMP2-DX2 wascloned into pcDNA3.1-myc. First, AIMP2-DX2 from H322 cDNA was amplifiedusing primers with linker for EcoRI and XhoI and cloned intopcDNA3.1-myc (Invitrogen) using EcoRI and XhoI. ThepcDNA3.1-myc/AIMP2-DX2 vector to express AIMP2DX (FIG. 12) wasintroduced into E. coli DH5α, which was deposited on Oct. 25, 2004 inthe International Depository Authority, the Korean Collection for TypeCultures (KCTC) and was given accession number KCTC 10710BP. In theRECEIP IN THE CASE OF AN ORIGINAL DEPOSIT attached, while theidentification reference read as Escherichia coli DH5@/p38DX2, it shouldbe noted that p38DX2 is newly designated herein as AIMP2-DX2.

Lung Caner Formation

32 AIMP2^(+/−) mice (19 male and 13 female mice) and 25 AIMP2^(+/+) mice(14 male and 11 female mice) were used for experiment. We induced lungtumor through the intraperitoneal injection of chemical carcinogen,benzo-(α)-pyrene (BP, 100 mg/kg, Sigma) into AIMP2^(+/+) and AIMP2^(+/−)mice, and monitored the tumor formation in lung. As a control group, 3AIMP2^(+/+) (2 male and 1 female) and 5 AIMP2^(+/−) mice (4 male and 1female) were injected with bical solution (10% DMSO and 35% PEG 40 insaline). The mice treated were sacrificed at the indicated time andtheir lung tissues were fixed in formaldehyde and undergone H&E stainingas described in Kim, M. J. et al., Nat. Genet. 34:330-336(2003),followed by the observation under a microscope.

Results

Functional Importance and Working Mode of AIMP2 in TGF-β Signaling

To see the importance of AIMP2 in TGF-β signaling, we compared theresponses of the AIMP2^(+/+) and AIMP2^(−/−) MEFs to TGF-β-inducedgrowth arrest, nuclear translocation of Smad2/3 and interaction of AIMP2with Smad2/3. While the growth of the wild type cells was suppressed byTGF-β, the AIMP2-deficient cells did not respond to the signal asdetermined by thymidine incorporation, colony formation and flowcytometry (FIGS. 1 a, 1 b and 1 c, respectively). When MEFs were treatedwith TGF-β, Smad2 and Smad3 were translocated to nuclei in the normalcells, but not in the AIMP2^(−/−) cells (FIG. 1 d). All of these resultssuggest the functional importance of AIMP2 in TGF-β signaling via Smad2and Smad3.

We then checked the possible interaction of AIMP2 with Smad2 and 3 bycoimmunoprecipitation. AIMP2 showed the interaction with Smad2/3 thatwas enhanced by TGF-β (FIG. 1 e). The direct interaction of AIMP2 withtwo R-Smads was also confirmed by yeast two hybrid and in vitropull-down assays (FIGS. 2 and 7). The amount of AIMP2 bound to Smad2/3was increased according to the induction of AIMP2 by TGF-β (FIG. 2 a).When the AIMP2 level was increased by transfection, the expression ofthe TGF-β target genes was enhanced, suggesting its stimulatory role inTGF-β signaling (FIG. 2 b). Since AIMP2 binds to both of Smad2 and 3, weexpected that it would work to these two R-Smads in a similar way andthus focused on its relationship to Smad2 in more detail. TheTGF-β-dependent phosphorylation of Smad2 was suppressed in theAIMP2-deficient MEFs, but restored when AIMP2 was introduced toAIMP2^(−/−) cells (FIG. 2 c). We then determined the domain of Smad2involved in the interaction with AIMP2 by yeast two hybrid (FIG. 2 d)and in vitro pull-down assay (FIG. 7). The two experiments revealed thatthe interaction involves the MH2 domain of Smad2.

To determine the working mode of AIMP2 in TGF-β signaling, we comparedthe TGF-β-induced association of Smad2 with TGF-β receptor in the normaland AIMP2-deficient cells. The cells were treated with TGF-β and theassociation of Smad2 and the receptor was monitored byco-immunoprecipitation of type I receptor with Smad2 at time interval.While the TGF-β-induced Smad2 binding to TGF-β receptor was observed atearly time point and decreased in the wild type cells, the receptorbound to Smad2 was accumulated in the late stage after TGF-β treatmentin the AIMP2-deficient cells (FIG. 2 e). In the TGF-β-inducedphosphorylation of Smad2, the phosphorylated Smad2 was graduallyincreased in the wild type cells, but severely suppressed in theAIMP2^(−/−) cells (FIG. 2 f). These results demonstrate that AIMP2 playsa critical role in the TGF-β induced phosphorylation of R-Smads via itsdirect interaction with R-Smad2.

Suppression of AIMP2 and Generation of its Deletion Variant in CancerCells

To explore the possible association of AIMP2 with cancer formation, wechecked the variation of AIMP2 level in different cancer cell lines[A549 (lung epithelial carcinoma), DU145 (prostate carcinoma), HCT116(human colorectal carcinoma), H460 (large cell lung carcinoma), H322(lung bronchioalveolar carcinoma) and H290 (mesothelioma cancer cellline) available from ATCC]. Three of the six tested cell lines showedlower AIMP2 level in Western blot (FIG. 3 a) and FACS analyses (FIG. 3b). All of the cell lines with low AIMP2 level expressed the normallevel of the TGF-β type II receptor and retained its kinase activity,implying that the low AIMP2 level does not result from themal-functionality of the receptor. To determine whether the variation ofAIMP2 level resulted from the difference in transcription, we performedRT-PCR with different combinations of the AIMP2-specific primers. TheAIMP2 gene consists of four exons (FIG. 8 a). When the primers were usedto generate AIMP2 cDNA spanning exon 3 and 4, the decrease of AIMP2transcript was not observed in the cells showing the reduced level ofAIMP2 (FIG. 3 c, first row), suggesting that it does not result fromlower transcription. When we used the primers generating the transcriptfrom exon 1 to 3, we obtained not only the transcript of the expectedsize, but also a smaller one (FIG. 3 c, second row). Sequencing analysisof this small transcript revealed that it lacks exon 2 encoding 69 aa ofAIMP2 (FIG. 8 b). To confirm the generation of this smaller transcript,we designed the primer (FIG. 8 b, primer DX-B) targeting to the junctionsequence of exon 1 and 3 that is generated by the deletion of exon 2,and conducted RT-PCR with this primer. The cell lines expressing lowerAIMP2 level generated the smaller transcript (designated AIMP2-DX2, FIG.3 c, second and third rows).

Western blot analysis with anti-AIMP2 antibody detected only thefull-length AIMP2, but not AIMP2-DX2 (FIG. 2 a), implying that AIMP2-DX2may be very unstable at protein level. Immunofluorescence staining alsodemonstrated the lower AIMP2 level in H460, H322 and H290 (FIG. 3 d). Toexclude the possibility of staining artifact, we co-cultivated H460 andDU145 cells in the same plate and stained AIMP2. Again, the stainingintensity of AIMP2 in H460 was much weaker than that in DU145 (FIG. 9a). In addition, the TGF-β-dependent nuclear localization of AIMP2 wasnot observed in the cells with low AIMP2 expression (FIG. 9 d, bottomrow). To address the linkage between the AIMP2 level and functionalityof TGF-β, we measured growth suppression by TGF-β in these cell lines.While the growth of A549 and DU145 cells was suppressed by TGF-β, thecells with low AIMP2 level did not respond to TGF-β (FIG. 9 e). Thisresult is consistent with previous reports that A549 is sensitivewhereas H460 is resistant to TGF-β signal (Osada, H. et al. Cancer Res.61, 8331-8339(2001); Kim, T. K. et al., Lung Cancer 31, 181-191(2001)).In addition, the target genes were induced by TGF-β in A549 and DU145,but not in H322 and two other cell lines with low AIMP2 (FIG. 3 f).

Furthermore, we tested by RT-PCR whether other cancer cell linesgenerates AIMP2-DX2. As a result, it was revealed that AIMP2-DX2 wasdetected in SaOS2 (osteosarcoma) and MCF7 (breast adenocarcinoma cell).

The Inactive Deletion Variant Forms a Complex with Functional AIMP2

To comprehend the causal relationship of the AIMP2-DX2 generation andsuppression of AIMP2, we checked the change of the AIMP2 level aftertransfection of AIMP2-DX2 into DU145. AIMP2 was reduced in theAIMP2-DX2-transfected cells, as demonstrated by Western blot (FIG. 4 a,first row) and FACS analyses (FIG. 9 b). Moreover, the expression of theAIMP2 target, c-myc, was elevated by the introduction of AIMP2-DX2 (FIG.4 a, third row). AIMP2-DX2 also relieved the growth arrest by TGF-β(FIGS. 4 b and 9 c). We then investigated how AIMP2-DX2 would affect thefunctional AIMP2 (AIMP2-F). Since AIMP2 has a potential to form ahomodimer (Quevillon, S. et al., J. Mol. Biol. 285, 183-195(1999); andKim, J. Y. et al., Proc. Natl. Acad. Sci. USA 99, 7912-7916(2002)), weexamined whether AIMP2-DX2 would interact with AIMP2-F by yeast twohybrid assay. AIMP2-DX2 showed the interaction with AIMP2-F, but notwith itself (FIG. 4 c). In in vitro pull-down assay, both ofradioactively synthesized AIMP2-F and AIMP2-DX2, but not AIMP2-DX2, wereco-purified with GST-AIMP2-F (FIG. 4 d), proving the direct interactionbetween AIMP2-F and AIMP2-DX2. To see whether AIMP2-DX2 is active inTGF-β signaling, we tested its interaction with Smad2 by yeast twohybrid assay. While AIMP2 interacted with Smad2 as well as FBP that isthe known target of AIMP2 (Kim, M. J. et al., Nat. Genet. 34,330-336(2003)), AIMP2-DX2 did not bind to any of these proteins,suggesting that it would be functionally inactive (FIG. 4 e).

To understand how the heterodimer formation would suppress AIMP2, wechecked whether the AIMP2 level is controlled by proteasome-dependentdegradation process. The AIMP2-DX2-producing H322 cells were treatedwith the proteasome inhibitor, ALLN (Zhou, M., et al., J. Biol. Chem.271, 24769-24775(1996)) and tested whether the AIMP2 level is increasedby the blockage of proteasome. The AIMP2 level was significantlyincreased by the treatment of ALLN as shown by Western blotting (FIG. 4f), immunofluorescence staining (FIG. 4 g) and flow cytometry (FIG. 9d), suggesting that its cellular level would be controlled byproteasome-mediated degradation. In addition, AIMP2-DX2 form was alsodetected by the inhibition of proteasome (FIG. 4 f), confirming thenotion that AIMP2-DX2 would be unstable due to the rapidproteasome-dependent degradation. The low intensity of AIMP2-DX2 appearsto result from its lower transcription compared to the normal AIMP2, asdemonstrated by RT-PCR analysis (FIG. 3 c) and/or less efficientrecognition by anti-AIMP2 antibody. Since AIMP2 degradation is mediatedby proteasome, we tested whether its ubiquitination is promoted byAIMP2-DX2. When AIMP2 was immunoprecipitated from the control andAIMP2-DX2-transfected cells that were treated with ALLN and blotted withanti-ubiquitin antibody, higher amount of the ubiquitinated AIMP2 wasobserved in the AIMP2-DX2-transfected cells compared to that in controlcells (FIG. 4 h). Combined together, AIMP2-DX2 appears to work as adominant negative mutant to form an inactive complex with AIMP2 that israpidly driven to degradation process.

AIMP2-DX2 Inactivates TGF-α Signaling and Promotes Cell Growth

We transfected AIMP2-DX2 into MEFs and monitored its effect on cellgrowth by microscopic analysis and colony formation. The cell growth wassignificantly enhanced by the introduction of AIMP2-DX2 (FIG. 5 a). Wethen introduced siRNA (si-AIMP2-DX2) that specifically suppresses theAIMP2-DX2 transcript and checked whether it can restore the normal levelof AIMP2 and TGF-β signaling in H322 cells expressing AIMP2-DX2.si-AIMP2-DX2 ablated the AIMP2-DX2 transcript (FIG. 5 b top) and resumedthe normal AIMP2 level and TGF-β-induced phosphorylation of Smad2 (FIG.5 b bottom), nuclear localization of p-Smad2 (FIG. 5 c), TGF-β-dependentreporter expression (FIG. 5 d) and growth arrest (FIG. 5 e). All ofthese results demonstrated the disruptive effect of AIMP2-DX2 on TGF-βsignaling and cell growth control.

Association of AIMP2 Deletion Variant with Human Lung Cancer

Since the reduction of AIMP2 is frequently detected in different cancercell lines (FIGS. 3 a, 3 b and 3 d), and loss of AIMP2 leads tohyper-proliferation of lung cells (Kim, M. J. et al., Nat. Genet. 34,330-336(2003)), we examined the association of AIMP2 abnormality withlung cancer formation. We induced lung tumor through the intraperitonealinjection of chemical carcinogen, benzo-(α)-pyrene (BP, Wang, Y. et al.,Cancer Res. 63, 4389-4395 (2003)) into AIMP2^(+/+) and AIMP2^(+/−) mice,and monitored the tumor formation in lung. From 6 weeks after theadministration of BP, lung tumors were observed at 50-70% frequency inAIMP2^(+/−) mice, and at about 30% in the wild type littermates (FIG. 6a), implying that the heterozygous mice are more susceptible toBP-induced tumorigenesis. We examined whether AIMP2-DX2 is generated inthe lungs of the BP-injected mice. Three out of four lungs isolated fromAIMP2^(+/−) mice showed tumors, while only one developed tumors amongthree AIMP2^(+/+) mice and all of these tumors generated AIMP2-DX2 (FIG.10), further supporting the relevance of AIMP2-DX2 to tumor formation.Moreover, AIMP2-DX2 was generated only in tumor tissues (FIG. 6 b). Toexclude the possibility that the AIMP2 level and AIMP2-DX2 formation mayvary depending on different individuals, we carried out RT-PCR analysesin the normal and tumor pairs isolated from the same patients. Again,the cancer-specific reduction of AIMP2 was coupled with the generationof AIMP2-DX2 (FIG. 6 c). AIMP2-DX2 was not detected in one case showingthe normal AIMP2 level in cancer region (FIG. 6 c, patient 22872). Wefurther examined 10 different pathologically-diagnosed lungadenocarcinoma, squamous cell carcinoma and large cell adenocarcinomasamples, and observed the cancer-specific reduction of AIMP2 andgeneration of AIMP2-DX2 in 8 cases (Table 1). Although AIMP2-DX2 wasdetected in the histologically normal regions in two cases, itsoccurrence was still coupled with the low level of AIMP2.

TABLE 1 The relationship between AIMP2 level and AIMP2-DX2 generation indifferent lung cancer patients Code Cell AIMP2-F AIMP2-DX2 No (MLLG)type Opdate Recur Sex Age N T N T 1 H004 SQC Jan. 06, 1926 FALSE M 64 +− − + 2 H008 ADC Jan. 09, 1926 FALSE M 62 + − − + 3 H001 ADC Jan. 12,1914 FALSE M 68 + − − + 4 H010 SQC Jan. 12, 1926 FALSE M 56 + − − + 5H018 ADC Feb. 04, 1926 FALSE M 60 − − + + 6 H021 ADC Feb. 05, 1907 TRUEF 64 − − + + 7 H024 ADC Feb. 05, 1917 FALSE F 59 + − − + 8 H025 SQC Feb.05, 1928 FALSE M 73 + − − + 9 H029 ADC Feb. 06, 1914 FALSE M 67 + − − +10 H031 LAC Feb. 07, 1908 FALSE M 71 + − − + Positive (+) and negative(−) in the AIMP2-F column denote the immunofluorescence staining ofAIMP2 in normal (N) and tumor (T) tissues determined by histologicalanalysis. Positive and negative in AIMP2-DX2 column indicate thegeneration of AIMP2-DX2 that was determined by RT-PCR. Note forabbreviations: Recur (recurrence), SQC (squamous cell carcinoma), ADC(adenocarcinoma), LAC (large cell adenocarcinoma) and Opdate (operationdate).Association of AIMP2 and its Deletion Variant with Human Liver Cancer

We examined the relationship of AIMP2 or its deletion variant with humanliver cancer using human tissues. The formation of AIMP2-DX2 wasevaluated in normal and cancer tissue (hepatocellular carcinoma) byRT-PCR and the level of AIMP2 by immunofluorescence analyses asdescribed previously. As a result, AIMP2-DX2 was detected in live cancertissues (FIG. 11).

Having described a preferred embodiment of the present invention, it isto be understood that variants and modifications thereof falling withinthe spirit of the invention may become apparent to those skilled in thisart, and the scope of this invention is to be determined by appendedclaims and their equivalents.

1. An isolated AIMP2-DX2 protein comprising the amino acid sequence ofSEQ ID NO:2.
 2. A method of screening for an agent which inhibits theformation of a heterodimer between the AIMP2-DX2 protein and the AIMP2protein, comprising the steps of: (a) contacting a test substance to acomposition which comprises the AIMP2-DX2 protein of SEQ ID NO:2 and theAIMP2 protein; and (b) determining whether the test substance inhibitsthe heterodimer formation between the AIMP2-DX2 protein and the AIMP2protein, wherein the test substance to inhibit the heterodimer formationbetween the AIMP2-DX2 protein and the AIMP2 protein is evaluated as ananticancer agent.
 3. The method according to claim 2, wherein the methodis performed by a yeast-two-hybrid assay or in vitro pull-down assay.